A New Natural Autotetraploid and Chromosomal Characteristics of Dwarf Snakehead Fish, Channa Gachua (Perciformes, Channidae) in Thailand
Total Page：16
File Type：pdf, Size：1020Kb
© 2014 The Japan Mendel Society Cytologia 79(1): 15–27 A New Natural Autotetraploid and Chromosomal Characteristics of Dwarf Snakehead Fish, Channa gachua (Perciformes, Channidae) in Thailand Alongklod Tanomtong1*, Weerayuth Supiwong1, Pornpimol Jearranaiprepame1, Suthip Khakhong2, Chanyut Kongpironchuen3, and Nuntaporn Getlekha1 1 Genetics and Environmental Toxicology Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Aquaculture Program, Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Muang 83000, Thailand 3 Faculty of Agriculture and Natural Resource, Rajamangala University of Technology Tawan-Ok, Sriracha, Chonburi 20110, Thailand Received October 15, 2012; accepted April 2, 2013 Summary A new natural autotetraploid and the chromosomal characteristics of the dwarf snakehead fish (Channa gachua) from Kalasin, Nong Khai, Nong Bua Lam Phu and Udon Thani Provinces (four populations) in northeast Thailand were studied. Kidney cell samples were taken from 12 male and 12 female fish. Mitotic chromosome preparations were conducted using a blood cell culture technique as well as taken directly from kidney cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the autotetraploid chromosome number of C. gachua was 4n = 104, and the fundamental number (NF) was 112 in both sexes. The types of chromosomes were 4 large submetacentric, 2 large acrocentric, 16 large telocentric, 2 medium submetacentric, 68 medium telocentric and 12 small telocentric chromosomes. No strange-sized chromosomes related to sex were observed. The region adjacent to the centromere of chromosome pair 3 showed clearly observable secondary constriction (NORs). The karyotype formula for C. gachua is as follows: sm a t sm t t 4n (autotetraploid) 104 = L4 +L2+L16+M2 +M68+S12 Key words Dwarf snakehead fish, Channa gachua, Autotetraploid, Ag-NOR staining, Chromosome. The study of fish and other aquatic animal chromosomes has become an active area of research in recent decades (Thorgaard and Disney 1990). Karyological studies have provided basic information on the diploid chromosome number (2n), fundamental number (NF), sex control and morphology of chromosomes (Khan et al. 2000, Tan et al. 2004). The information may be useful for addressing a variety of evolutionary and genetic questions about animals (MacGregor 1993) and may permit detection of changes that modified an ancestral karyotype as it evolved into new lines (Winkler et al. 2004, Kalbassi et al. 2006). These studies are also an important step towards the establishment of genetic improvement techniques involved in chromosome manipulation techniques, including inter- or intra-species hybridizations, sex control, gynogenesis, androgenesis and induction of polyploidy (Wu et al. 1986, Diter et al. 1993). These genetic techniques have been widely applied to improve farmed stocks in many aquaculture species in the world (Arai 2001, Beardmore et al. 2001, Dunham 2007, Chai et al. 2009). The dwarf snakehead fish, Channa gachua (Hamilton 1822), is a member of class * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.79.15 16 A. Tanomtong et al. Cytologia 79(1) Actinopterygii, order Perciformes and family Channidae. At the present, there are two genera and 29 species in the family Channidae. Seven species of channid fish have been found in Thailand (Courtenay and Williams 2007, Vidthayanon 2005). Only six species have been studied cytogenetically, each having a different diploid number: C. marulioides, 2n = 38 (Magtoon et al. 2006); C. striata, 2n = 42 from Northeastern Thailand (Supiwong et al. 2009), 2n = 44 from Central Thailand (Wattanodorn et al. 1985, Donsakul and Magtoon 1991); C. micropeltes, 2n = 44 (Donsakul and Magtoon 1991, Supiwong and Jearranaiprepame 2009); C. marulius, 2n = 44; C. lucius, 2n = 48 and C. gachua, 2n = 112 (Donsakul and Magtoon 1991). Variations in chromosome number and morphology can be found among C. striata populations in Thailand (Supiwong et al. 2009). The C. gachua has been found in Asian countries from Pakistan to Indonesia. Modern ichthyologists consider this fish to be a species complex, i.e. it consists of several closely related species. Because of its pretty coloration and small size, it is an attractive fish and commonly kept in aquaria. It is a mouth brooder and feeds on a wide range of foods including insects and smaller fish. It is a hardy fish and can tolerate changes in temperature and acidity well. The C. gachua is often confused with C. orientalis, an endemic species from Sri Lanka. In Indian ichthyology, C. gachua is considered to be a junior synonym of C. orientalis as it was described first by Markus Elieser Bloch, then 20 years later Hamilton reported that C. gachua has ventral fins while C. orientalis has none. Furthermore, there are many other differences, including their breeding behaviour and number of offspring (Courtenay and Williams 2007). The objective of this study was to investigate the karyotype analysis and chromosomal characteristic of NORs in the C. gachua from Thailand. The study would also improve our understanding of karyotype evolution mechanism, speciation in the genus Channa, and increase knowledge available for implementation of polyploidy manipulation, hybridization, sex control and other genetic improvements in the future. Materials and methods Sample collection The samples of C. gachua (12 males and 12 females) were obtained from Kalasin, Nong Khai, Nong Bua Lam Phu and Udon Thani Provinces (four populations) in Northeastern Thailand. The fish were transferred to laboratory aquaria and kept under standard conditions for 7 d prior to the experiments. Chromosome preparation Chromosomes were prepared in vivo (Chen and Ehbeling 1968, Nanda et al. 1995) as follows. Phytohemagglutinin (PHA) solution was injected into the fish abdominal cavity. After a 24 h period, colchicines were injected into the fish’s intramuscular and/or abdominal cavity and then left for 2–4 h. The kidney, spleen and/or gills were cut into small pieces and then squash mixed with 0.075 M KCl. After discarding all large pieces of tissue, 15 mL of cell sediments was transferred to a centrifuge tube and incubated for 25–35 min. KCl was discarded from the supernatant after being centrifuged at 1,200 rpm for 8 min. The cells were fixed in a fresh, cool fixative (3 methanol : 1 glacial acetic acid) of which up to 8 mL were gradually added before being centrifuged again at 1,200 rpm for 8 min, at which time the supernatant was discarded. The fixation was repeated until the supernatant was clear and the pellet was mixed with 1 mL of fixative. The mixture was dropped onto a clean and cold slide by a micropipette, followed by the air-dry technique. Chromosome staining Conventional staining was performed using 20% Giemsa’s solution for 30 min, and Ag-NOR 2014 A New Natural Autotetraploid and Chromosomal Characteristics of Channa gachua 17 banding (Howell and Black 1980) was done by adding two drops of 50% silver nitrate and 2% gelatin on slides. The slides were then sealed with cover glasses and incubated at 60°C for 5 min. After that they were soaked in distilled water until the cover glasses were separated. The slides were stained with 20% Giemsa’s solution for 1 min. Chromosome checks Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well-spread chromosomes of each male and female were selected and photographed. The length of the short arm chromosome (Ls) and the length of the long arm chromosome (Ll) were measured, and the length of the total arm chromosome (LT, LT = Ls+Ll) was calculated. The relative length (RL), centromeric index (CI) and standard deviation (SD) of RL and CI were estimated (Chaiyasut 1989). The CI (q/p+q) between 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90–0.99 were described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. The fundamental number (number of chromosome arms, NF) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes, and a value of one to telocentric chromosome. All parameters were used in karyotyping and idiograming. Results and discussion Chromosome number, fundamental number and karyotype of C. gachua The analysis of 200 metaphase spreads from 12 males and 12 females of C. gachua (four populations) clearly demonstrates that the model chromosome number in this species is tetraploid (4n) = 104 in both sexes (Fig. 1). This is different from previous studies by Donsakul and Magtoon (1991), which have shown 2n = 112 for C. gachua in Thailand, and by Banerjee et al. (1988), which reported 2n = 78 in India. Variations on chromosome number and morphology can be found among different populations, including those of C. striata in Thailand (Supiwong et al. 2009) and C. asiatica in China (Yu and Zhou 1996). Furthermore, interpopulational chromosome variation has also been observed in the Erythrinid fish, Hoplerythrinus unitaeniatus (Diniz and Bertollo 2003, Giuliano-Caetano et al. 2001). This study found that the genome of C. gachua results from polyploidy of two genome-doubling (autotetraploid, 4n). It is consistent with the study of Rishi and Haobam (1990) who reported the autotetraploid (4n) = 104 of C. stewartii, which belong to the same genus, Channa, as C. gachua. Figure 3 shows the idiogram of the C. gachua from conventional staining. Two genome-doubling events are considered to have taken place before the tetraploids split from the fish 360 million years ago (Allendorf and Thorgaard 1984, Wittbrodt et al. 1998, Zhou et al. 2002), resulting in an ancestral diploid chromosome number (2n) of approximately 48. While uncommon in higher vertebrates, polyploidy has appeared repeatedly during the development and diversification of fishes, from sharks to higher teleosts. Many economically important fish, such as carp, salmon and sturgeon, have evolved from polyploidy ancestors. Additionally, artificially- induced polyploidy has been used in aquaculture to induce sterility or improve production (Donaldson and Devlin 1996).